Mixed function oxidation. V. Flavin interaction with a reduced diphosphopyridine nucleotide dehydrogenase, one of the enzymes participating in camphor lactonization.

3. One mole of the apoenzyme, molecular weight 36,000, will bind 1 mole of flavin mononucleotide, 2 moles of flavin adenine dinucleotide, or 1 of each when presented with them at equal concentrations. Two flavin-binding sites are present; one is common to both flavins and is catalytically active. The other is specific for flavin adenine dinucleotide and is not involved in catalysis. Equal concentrations of these two flavins are present in crude cell extracts; riboflavin is absent. 4. The dissociation constant, K,, for the “apoenzymeflavin mononucleotide” complex is 4.5 X 10-T M. The minimum turnover number for reduction of the complex by reduced phosphopyridine nucleotide is 30,000. Michaelis constants, K,, for interaction of free flavin mononucleotide and reduced diphosphopyridine nucleotide with the complex are 3 X 10-6~ and lo-* M, respectively. 5. The dissociation constants for the “apoenzyme-2 flavin adenine dinucleotide” complex lie within the range 2.5 to 7 X 1OF M. A minimum turnover number for reduction of this complex is 39,000. Michaelis constants for interaction of free flavin adenine dinucleotide and reduced diphosphopyridine nucleotide with the complex are 1.9 X 10e5 M and 2.1 X 10-4 M, respectively. 6. Sulfhydryl groups and iron are not involved in flavin or reduced diphosphopyridine nucleotide binding; reduced diphosphopyridine nucleotide is not bound in the absence of flavin. Oxidation and reduction of an enzyme sulfhydryl group are not involved in catalysis. 7. Reduction of the “apoenzyme-flavin mononucleotide” complex proceeds by two l-electron transfer steps. The first, more rapid transfer yields the flavin semiquinone. Re-